Leptospirosis is a globally distributed zoonotic disease caused by pathogenic Leptospira spp., which are classified into numerous serogroups. Accurate serogroup identification is essential for epidemiological understanding and public health interventions in endemic regions. The microscopic agglutination test (MAT) is the conventional serological method for serogroup identification; however, it is labor-intensive and depends on extensive reference strain panels. Molecular serogroup typing (MST) has been proposed as an alternative approach, but its performance for region-specific strains has not been sufficiently evaluated. In this study, we improved an MST method optimized for Japanese Leptospira isolates and applied it to determine the serogroups of pathogenic Leptospira spp. detected in kidneys of wild boars (Sus scrofa) and sika deer (Cervus nippon) collected in Yamaguchi Prefecture, Japan, between 2016 and 2025. Pathogenic Leptospira DNA was detected in 23 of the 190 wild boars (12.1%) and 22 of the 275 sika deer (8.0%) by real-time PCR targeting lipL32. The improved MST identified serogroups Autumnalis, Australis, Canicola, and Hebdomadis in both host species, with Autumnalis predominating. In contrast, MAT using standard reference strains detected antibodies mainly against Hebdomadis and Australis, indicating discrepancies in the predominant serogroups between molecular and serological results. Notably, MAT using a local Autumnalis strain successfully detected Autumnalis antibodies in both hosts. These findings demonstrate that the improved MST complements serological testing.

Background The gut microbiota of bats is increasingly studied for its ecological and public health significance, yet factors shaping gut microbial communities, such as host species, environmental location, and ecological variation, remain poorly understood in insectivorous bats in Japan. We characterised the gut microbiota of three insectivorous bat species ( Miniopterus fuliginosus , Myotis macrodactylus , and Rhinolophus ferrumequinum ) collected from three sites in Yamaguchi Prefecture, using 16S rRNA gene (V3–V4) High-throughput amplicon sequencing. We further screened for pathogenic bacteria using species-specific nested PCR. Results The gut microbiota was dominated by Pseudomonadota (63.6%) and Bacillota (22.3%). A global core of ten genera accounted for 74.5% of total relative abundance across all three bat species. Nested PCR detected DNA of Chlamydophila psittaci , Bacillus cereus , and Shiga toxin 1-producing Escherichia coli . PERMANOVA revealed that both host species (R² = 0.166, p = 0.001) and sampling location (R² = 0.132, p = 0.001) significantly influenced community composition based on Bray-Curtis dissimilarity, while alpha diversity did not differ significantly among species or locations. Functional profiles predicted by PICRUSt2 showed no significant differentiation among species ( p = 0.114) or locations ( p = 0.412), suggesting potential functional redundancy despite taxonomic variation. Co-occurrence network analysis revealed species- and location-specific patterns in the connectivity of core and potentially pathogenic taxa. Conclusions This study provides the first detection of Chlamydophila psittaci DNA in bats, alongside other pathogenic species, Bacillus cereus and Shiga toxin-producing Escherichia coli . In addition, we provide a comprehensive characterisation of the gut microbiome of insectivorous bats in Japan and show that both host species and sampling location significantly influence gut community composition. Meanwhile, predicted metabolic functions remained conserved, consistent with potential functional redundancy. The detection of pathogenic bacteria underscores the value of integrating wildlife microbiome surveillance into One Health frameworks. Future studies incorporating culture-based methods and whole-genome sequencing would strengthen the assessment of zoonotic potential.

Tick-borne viruses (TBVs) include the highly pathogenic viruses associated with infectious diseases in both humans and animals. The global public health concern of TBVs is closely related to geographic distribution of ticks. In Africa, a variety of TBVs have been reported by detections from ticks and high seroprevalence in humans and domestic animals. In this study, we aimed to detect TBVs from ticks in Kenya by targeting viral genes of Orthoflavivirus, Bandavirus and Orthonairovirus. Ticks were collected from cattle, camels, sheep and goats in Isiolo, Narok and Baringo counties, and viral RNA detections were attempted by RT-PCR. There were no positive samples for Orthoflavivirus and Bandavirus. On the other hand, 10 samples tested positive for Orthonairovirus by RT-PCR, were identified from Rhipicephalus appendiculatus tick pools. Five pools were indicated as novel Orthonairovirus species, marigat tick orthonairovirus (MTOV). Interestingly, other 5 samples were considered to be Bole tick virus 4 (BLTV4) and BLTV4-related virus, marigat tick flavivirus (MTFV), belonging to unclassified genus within family Flaviviridae, but not Orthonairovirus. Our findings add to the knowledge of genetic diversity and geographic distribution of tick-borne viruses including novel Orthonairovirus MTOV, unclassified Flavivirus BLTV4 and MTFV in Kenya, and will provide useful clues for further epidemiological surveys.

The skin harbors a complex immune microenvironment that integrates innate and adaptive components and plays a critical role in vector-borne pathogen transmission. To investigate immune responses during tick-mediated transmission of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), we analyzed skin from BALB/c mice bitten by artificially infected Amblyomma testudinarium nymphs. Skin samples were collected at time points corresponding to peak viral RNA levels (days 6-7 post-attachment) and analyzed by transcriptomics, followed by real-time PCR and immunohistochemical (IHC) analyses. Transcriptomic analysis revealed increased expression of immune-related genes, particularly those associated with type I interferon signaling. Interferon-stimulated genes (e.g., Rsad2, Ifit family members) and monocyte-associated markers (e.g., Ly6c2) were elevated in RNA sequencing data, while real-time PCR analysis showed similar trends but did not reach statistical significance. IHC revealed colocalization of viral antigens with macrophages in the infected skin, as characterized by Iba1 expression. Further analysis demonstrated a higher density of Ly6c2-positive inflammatory monocytes, consistent with transcriptomic observations. In addition, BST2 (Tetherin) expression was increased, consistent with activation of a localized Type I IFN-mediated antiviral response. However, the strong inflammatory response induced by tick feeding may obscure infection-specific transcriptional changes at these later time points. Overall, our findings demonstrate that tick-mediated SFTSV infection was associated with interferon-related gene expression and recruitment of monocyte-lineage cells at the skin interface, while highlighting the complex interplay between viral infection and tick-induced host responses.

In Japan, Lyme disease and tick-borne encephalitis (TBE) are primarily in the northernmost prefecture Hokkaido, where their primary vectors Ixodes ovatus and Ixodes persulcatus are most abundant. Recently, tick surveillance activities have collected both species across Japan, indicating potential expansion of their tick-borne pathogens (TBPs). We built a machine-learning (ML) model using available tick surveillance and environmental data to predict suitable habitat areas for I. ovatus and I. persulcatus and identify potential higher-risk areas of Lyme disease and TBE across Japan. Data on the occurrence and abundance of 11 vector tick species between 1990 and 2023 were extracted from studies identified via systematic literature search in two online databases or provided by local experts for ML development. After multiple iterations and permutations, separate Random Forest ML algorithms for I. ovatus and I. persulcatus were trained via 26 abiotic variables of climate and topography based on the occurrence and abundance respective to each species. Data on 93,289 ticks from 57 sources were extracted, and the ML algorithms' area under the curves were high (> 0.89). Climate-related variables were the strongest predictors (> 90% cumulative model importance) for both I. ovatus and I. persulcatus. High suitability for both species was identified in Hokkaido and cooler, wetter regions in central Honshu, while I. ovatus had a broader ecological niche than I. persulcatus, with moderate suitability in mountainous regions of central Kyushu and surrounding the Tokyo Bay area. Our ML models suggest high suitability areas for Ixodes vectors may be widespread in Japan, indicating expanding potential risks of TBPs.

Leptospirosis is a zoonotic infectious disease caused by pathogenic Leptospira species. In Japan, human and canine leptospirosis cases are reported annually. Although wild animals, including bats, are known to harbor pathogenic Leptospira spp., infection of bats in this region has never been explored. This study aimed to determine the presence of leptospires and their genetic diversity in five Japanese bat species. Kidney tissue and blood samples were collected from 115 bats captured in the Yamaguchi Prefecture and Hokkaido, Japan. Pathogenic Leptospira spp. were detected in the kidneys by real-time PCR and conventional PCR using primers targeting lipL32 and rrs2. Multilocus sequence typing (MLST) and sequencing of secY gene were performed on PCR-positive DNA samples. Leptospira spp. were detected in 26 of the 115 bats (22.6 %), including Rhinolophus ferrumequinum (5/37, 14 %), Rhinolophus cornutus (2/6, 33 %), Miniopterus schreibersii (6/37, 16 %), Myotis macrodactylus (11/15, 73 %), and Vespertilio sinensis (2/20, 10 %). Phylogenetic analysis based on MLST genes and secY gene revealed detected genes clustered with either L. interrogans, L. borgpetersenii, or L. kirschneri and a relationship between the leptospires identified in the bats in this study and bats in other countries or other host, and PCR with L. borgpetersenii-specific primers revealed co-infection with multiple Leptospira species in individual bats. Our study demonstrated a high carriage rate, genetic diversity of Leptospira spp., and co-infection with multiple Leptospira spp. in Japanese bats.

Borrelia miyamotoi is a hard tick-borne spirochete genetically related to relapsing fever Borrelia and the etiological agent of an emerging infectious disease in humans. Like relapsing fever Borrelia, B. miyamotoi carries clusters of gene cassettes encoding variable major proteins (Vmps) on multiple linear plasmids and shows antigenic variation in mammalian hosts by switching the expression vmp gene cassette. However, it remains unknown how the switch occurs in B. miyamotoi. Here we determined the whole genome sequences of Japanese B. miyamotoi strains to identify the repertoire and arrangement of vmp gene cassettes on five linear plasmids, and based on this information, analyzed B. miyamotoi clones reisolated from experimentally infected mice. Our analyses revealed that the switch occurred by replacing the expression cassette and its downstream silent cassettes with the long segment from archival plasmid. As the result of this long segment conversion, the first cassette became the expression cassette. Notably, this phenomenon was not due to single gene conversion but the replacement of a long (up to 16 kb or more) plasmid segment. We also show that while bacterial elimination depended on the presence of specific antibodies, the segment conversion was detected at five days post-infection, earlier than antibody production in mice, and even in severe combined immunodeficient mice. These results provide novel insights into the mechanisms that Borrelia evolved to survive and persist in mammalian hosts.

Background and Purpose: Clinical manifestations of Lyme borreliosis (LB), caused by Borrelia burgdorferi sensu lato (Bbsl), include erythema migrans, Lyme neuroborreliosis (LNB), carditis, and arthritis. LB is a notifiable disease in Japan with <30 surveillance-reported LB cases annually, predominately from Hokkaido Prefecture. However, LB, including LNB, may be under-diagnosed in Japan since diagnostic tests are not readily available. We sought to determine if LNB could be a cause of previously undiagnosed encephalitis or meningitis in Japan. Methods: Investigators at 15 hospitals in 10 prefectures throughout Japan retrieved serum and/or cerebrospinal fluid (CSF) samples collected in 2010-2021 from 517 patients hospitalized with encephalitis or meningitis which had an etiology that had not been determined. Samples were tested for Bbsl-specific antibodies using ELISA and Western blot tests. In alignment with the European Union LNB case definition, a confirmed LNB case had CSF pleocytosis and intrathecal production of Bbsl-specific antibodies and a probable LNB case had a CSF sample with pleocytosis and Bbsl-specific antibodies. Results: LNB was identified in three hospitalized patients with meningitis of previously undetermined etiology: a male resident of Aomori Prefecture was a confirmed LNB case, and two female residents of Oita Prefecture were probable LNB cases. None of the patients with confirmed or probable LNB had traveled in the month prior to symptom onset and none had samples previously tested for LB. Conclusion: The identification of previously undiagnosed LNB cases indicates a need for enhanced disease awareness in Japan, particularly beyond Hokkaido Island, and more readily available LB diagnostic testing.

The correct delineation of tick species is critical for efforts aimed at safeguarding One Health. Historically, the Asian turtle tick (Amblyomma geoemydae sensu lato) has been regarded as a geographically widespread species across much of Asia. However, based on morphological and phylogenomic data, the subtropical lineage (from Japan, Taiwan, and parts of China) of A. geoemydae is recognised as a new species: Amblyomma kappa sp. n., and all life stages are described. The tropical lineage of A. geoemydae sensu stricto is recharacterized and a differential diagnosis is provided to distinguish the two species. The ecology, host associations, and medical importance of A. kappa are also discussed.

Francisella tularensis is an intracellular gram-negative bacterium known as the causative agent of tularemia, which can be transmitted to humans by direct contact with wild animals or by tick bites. Although F. tularensis is highly pathogenic, its recent prevalence in Japan is underreported due to the small number of reported cases. To clarify the current situation of F. tularensis in wild animals, we conducted surveillance on various species of wild animals in Yamaguchi prefecture. In this study, we screened 809 samples collected from 90 Japanese black bears, 105 Japanese monkeys, 168 sika deer, 205 wild boars, and 84 bats. For seroprevalence analysis, we tested 177 serum samples from 75 black bears and 102 monkeys using the microagglutination test. The results showed that serums from five black bears exhibited slight agglutination. Western blot was performed as a confirmatory test on these five samples, but no positive signals were detected. Additionally, molecular surveillance was conducted using DNA extracted from 464 whole blood and 168 tissues, targeting the gene encoding 23 KDa hypothetical protein by real-time PCR and outer membrane protein A gene by conventional PCR. No positive samples of F. tularensis were detected by either real-time or conventional PCR. Although we did not detect any F. tularensis-positive samples through serological and molecular analyses, continuous surveillance studies are necessary since sporadic human cases have been reported in Japan.

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium transmitted by larval trombiculid mites, responsible for scrub typhus in humans. In Japan, approximately 500 human cases are reported annually, with six major serotypes identified as Kato, Karp, Gilliam, Irie/Kawasaki, Shimokoshi, and Hirano/Kuroki. However, the prevalence of OT in Yamaguchi prefecture remains largely unknown. This study aimed to investigate the prevalence and serotypes of OT in wild rodents collected from eight locations within the prefecture. DNA was extracted from spleen and liver samples of 135 wild rodents collected between 2015 and 2024, and PCR was conducted to detect OT targeting the 56 kDa type-specific antigen gene. As a result, five individuals (3.7%) were detected positive for OT DNA. OT was successfully isolated from two of the five rodents and characterized by multi-locus sequence analysis (MLSA) based on 11 housekeeping genes. The MLSA results indicated that both isolates clustered with OT strain Ikeda (JG serotype). Additionally, we performed a serological test on 117 serum samples from wild rodents using the indirect immune peroxidase test. The results showed that 59.8% (n = 70/117) of the rodents had antibodies against OT, with 73% (n = 51/70) showing the highest titer against the OT strains Gilliam (Gilliam serotype) and Ikeda (JG serotype), known to be transmitted by Leptotrombidum pallidum mites. Overall, the present study identified the OT serotypes and potential primary vector species in Yamaguchi prefecture, emphasizing the need for further surveillance, particularly, in humans.

Bats are important natural hosts of various zoonotic viruses, including Ebola virus, Lyssa virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). Although investigation of bats is valuable for predicting emerging infectious diseases from these animals, few surveys of bat-derived viruses have been conducted in Japan. In the present study, samples were collected from a total of 132 bats of 4 different species from 4 different locations within Yamaguchi Prefecture; these sample were employed for comprehensive detection of bat-derived viruses by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR using primers universal for each of 4 different viral classes. As a result of PCR and RT-PCR, various herpesviruses, astroviruses, coronaviruses, and adenoviruses were identified from a total of 80 bats. The detected herpesviruses belong to the Betaherpesvirinae or Gammaherpesvirinae subfamily, the detected adenoviruses to the genus Mastadenovirus, the detected astroviruses to the genus Mamastrovirus; and the detected coronaviruses belong to the genus Alphacoronavirus. The detected sequences of 12 strains of 4 families showed 100 % amino acid identity with viruses previously detected either in China or South Korea. These findings expand our understanding of viruses carried by bats, and provide insights into the nature of bat-derived viruses in Japan.

Migratory birds carry ticks harboring various pathogens, including the zoonotic Yezo virus. In Hokkaido, Japan, we collected ticks from migratory birds during 2020-2021. Eight of 385 pools, comprising 2,534 ticks, tested positive for Yezo virus RNA, suggesting Yezo virus might be spread through the flyways of migratory birds.

Historically, for more than one and a half centuries, only one so-called “long-legged bat tick” species, i.e., Ixodes vespertilionis Koch was known to science. However, during the past decade, it was recognized on a molecular basis that long-legged ixodid ticks associated with bats may represent at least six species. Of these, until recently, five have been morphologically described. In this study, Ixodes ticks were collected from two Myotis species in southeastern Asia, Vietnam. Based on the morphological and molecular characteristics of the female, nymph and larva, Ixodes lanigeri Hornok, sp. nov. is described here. The male is unknown. Like other members of the Ixodes ariadnae complex, I. lanigeri Hornok apparently shows a preference for vesper bats as its typical hosts. In this context, host-association and geographical separation may explain the evolutionary divergence of I. lanigeri Hornok from its closest relative occurring on Murina hilgendorfi Peters in East Asia, Japan, because no Myotis or Murina spp. have overlapping distribution between Vietnam and the main islands of Japan. On the other hand, supposing that (similarly to I. ariadnae) I. lanigeri Hornok probably occurs on other myotine bats and knowing that several Myotis species indigenous in Vietnam have a broad geographical range in southern and southeastern Asia, the new tick species most likely has a widespread distribution in this area.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

In Eurasia, the geographically most widespread ixodid tick species of the bat families Rhinolophidae Gray, Vespertilionidae Gray, and Miniopteridae Dobson were considered to belong to four species, Ixodes vespertilionis Koch, I. collaris Hornok, I. ariadnae Hornok, and I. simplex Neumann. Previous data attest that bat-associated tick species from Eastern Asia show remarkable genetic difference from the above four tick species, but in the absence of detailed morphological comparison these were regarded as conspecific. In this study we compensate for this lack of data on three bat-associated tick species, reporting their morphological comparison, as well as molecular and phylogenetic relationships. According to the results we describe the females of three tick species new to science, i.e., I. nipponrhinolophi Hornok & Takano, sp. nov., I. fuliginosus Hornok & Takano, sp. nov., and I. fujitai Hornok & Takano, sp. nov. In case of all three new tick species the cytochrome c oxidase subunit (coxI) gene showed remarkably high sequence differences from the species that they previously were thought to belong to, well exceeding the average limit delineating ixodid tick species. This, as well as observed morphological differences fully justify their taxonomical status as new species.

Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."

Background Borrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia , once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards ( Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia . Methods We used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus -associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date. Results The genomic architecture of Varanus -associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage. Conclusions In this work we present four new genomes from three novel Borrelia , and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii , ‘ Candidatus Borrelia undatumii’, and ‘ Candidatus Borrelia rubricentralis’ for them. Graphical Abstract